The research interests of my laboratory are centered on the molecular details of animal RNA virus replication and their interactions with vertebrate and invertebrate hosts. The experiments detailed in this proposal concern our current studies on Sindbis virus, type virus of the family Togaviridae. Several members of this family are agents of chronic arthropod-transmitted world health problems, and our long range goals include a thorough understanding of viral RNA replication and gene expression, virion assembly, and viral pathogenesis and immunity. We eventually hope to apply this information for design of antiviral therapies and effective vaccines. In addition, we hope to develop this virus system as a tool for studying broader questions relevant to human immunity and cell biology. Full-length cDNA clones of Sindbis virus capable of producing infectious RNA transcripts have made mapping causal mutations for biologically interesting phenotypes routine, and allow defined mutations and foreign sequences to be inserted into the viral genome to test a number of hypotheses concerning the functions of viral RNA sequences and proteins. Towards understanding the function of the nonstructural proteins (nsPs) in RNA replication, we are characterizing directed mutations in these proteins which result in lethal, conditional or deleterious phenotypes (and compensating reversions) as well as developing biochemical assays to study their function. Specific studies include: 1) The genetic identification of the protein(s) which interacts the promoter for subgenomic RNA transcription. 2) The role of nsP3 phosphorylation in RNA replication. 3) The production of nsPs in eucaryotic and procaryotic expression systems for functional in vitro and in vivo assays. The second major focus of this proposal is to pursue the use of Sindbis virus as an expression vector for heterologous RNAs and polypeptides. Random mutagenesis is being used to incorporate heterologous epitopes into Sindbis virus glycoproteins. Studies of such recombinants viruses in vivo will begin to address the question of whether or not engineered alphaviruses might be useful as protective immunogens. Novel replication and packaging competent Sindbis recombinants which express heterologous products via a second subgenomic RNA have also been constructed. In addition to being useful for production of defined products in animal or insect cells, these vectors can be used to study various aspects of the immune response to infectious agents or candidate vaccines, and to test the activity in vivo of trans-acting ribozymes designed to cleave specific cellular and viral RNA substrates. Such ribozymes, designed to cleave and inactive specific target RNAs, could be useful for studies of gene function and as antiviral agents.